Biomedical subjects

Q Xie

Publications and source records attributed to Q Xie.

At least 19 recordsLinked to original sources

PubMed2001

Ribozyme-mediated inhibition of caspase-3 activity reduces apoptosis induced by 6-hydroxydopamine in PC12 cells.

6-Hydroxydopamine (6-OHDA) is a neurotoxin used in the induction of experimental Parkinson's disease in both animals and PC12 cells, which are derived from rat pheochromocytoma tumors and have many properties similar to dopamine neurons. Biochemical and molecular approaches have shown that low doses of 6-OHDA induce apoptosis in PC12 cells and, in the processing of apoptosis, caspases are crucial mediators, and caspase inhibition is sufficient to rescue PC12 cells from apoptosis induced by 6-OHDA. However, because this caspase inhibition targets multiple caspases, it is not known whether a single caspase is primarily responsible for effecting cell death in this model. To assess the particular member (caspase-3) of the ced-3 family relevant to cell death and to position their activation within the apoptotic pathway, we constructed a hammerhead ribozyme directed against rat caspase-3, which could downregulate the expression of caspase-3 in vitro and in vivo, and transfer to PC12 cells. The results show that the ribozymes against caspase-3 could protect PC12 cells from apoptosis induced by low doses of 6-OHDA. The PC12 cell transfected with the ribozymes shows a significant decrease in caspase-3 activity compared with control cells at various time points. Parallel to the reduced caspase-3 protease activity, similar decreased levels of apoptotic cells and DNA fragmentation were also assessed by staining with Hoechst 33258 and ELISA, respectively. Overexpression of p35, a general caspase inhibitor, also protected PC12 cells from apoptosis. These results confirm that caspases play an important role in 6-OHDA-induced PC12 cell apoptosis and indicate that caspase-3 itself is one of the crucial mediators of neurotoxin-induced PC12 cell apoptosis.

Adrenergic Agents↗

PubMed2001

A piezoelectric quartz crystal impedance study on Cu(2+)-induced precipitation of bovine serum albumin in aqueous solution.

Piezoelectric quartz crystal impedance (QCI) technique was used for monitoring the Cu(2+)-induced precipitation of bovine serum albumin onto the gold electrode. The critical precipitate concentration of Cu(2+) reflected by the significant decrease in the resonant frequency was estimated to be 9.98 x 10(-5) mol x l(-1), and the saturated adherence of the precipitate on the electrode occurred when the Cu(2+) concentration was greater than 9.79x10(-3) mol x l(-1). The frequency shift in air was about 85.5% of that in liquid, and the Deltaf(0)/DeltaR(1) ratio found in solution was 82.67 Hz Omega(-1), suggesting that the frequency response was predominated by the mass change due to precipitate adherence to the electrode surface. The response of the resonant frequency was analyzed using an equation Deltaf=a(0) + a(1) e(-t/tau(1)) + a(2) e(-t/tau(2)). The relationship between the total a(0) values and the Cu(2+) concentration was discussed.

Animals↗

PubMed2001

Structure-activity relationships for a large diverse set of natural, synthetic, and environmental estrogens.

Understanding structural requirements for a chemical to exhibit estrogen receptor (ER) binding has been important in various fields. This knowledge has been directly and indirectly applied to design drugs for human estrogen replacement therapy, and to identify estrogenic endocrine disruptors. This paper reports structure-activity relationships (SARs) based on a total of 230 chemicals, including both natural and xenoestrogens. Activities were generated using a validated ER competitive binding assay, which covers a 10(6)-fold range. This study is focused on identification of structural commonalities among diverse ER ligands. It provides an overall picture of how xenoestrogens structurally resemble endogenous 17beta-estradiol (E(2)) and the synthetic estrogen diethylstilbestrol (DES). On the basis of SAR analysis, five distinguishing criteria were found to be essential for xenoestrogen activity, using E(2) as a template: (1) H-bonding ability of the phenolic ring mimicking the 3-OH, (2) H-bond donor mimicking the17beta-OH and O-O distance between 3- and 17beta-OH, (3) precise steric hydrophobic centers mimicking steric 7alpha- and 11beta-substituents, (4) hydrophobicity, and (5) a ring structure. The 3-position H-bonding ability of phenols is a significant requirement for ER binding. This contributes as both a H-bond donor and acceptor, although predominantly as a donor. However, the 17beta-OH contributes as a H-bond donor only. The precise space (the size and orientation) of steric hydrophobic bulk groups is as important as a 17beta-OH. Where a direct comparison can be made, strong estrogens tend to be more hydrophobic. A rigid ring structure favors ER binding. The knowledge derived from this study is rationalized into a set of hierarchical rules that will be useful in guidance for identification of potential estrogens.

Animals↗

PubMed2001

Development of a thickness shear mode acoustic sensor based on an electrosynthesized molecularly imprinted polymer using an underivatized amino acid as the template.

The preparation and characterization of electrosynthesized poly(o-phenylenediamine) (iPoPD) as a molecular imprinting material were studied by an in situ quartz crystal impedance method. The changes of delta f0, delta R1, delta L1 and delta C0 suggest that the polymer film was compact and rigid. The thickness shear mode (TSM) acoustic sensor modified with this material exhibits molecular recognition ability to the template molecule of DL-phenylalanine. In the range 2-20 mM, a linear relationship between the frequency shift delta f0 and logC was found from the calibration graph. Scatchard analysis of the relevant calibration graph offers information on the equilibrium of the binding interaction and the recognition sites. Using this electropolymerization technology, the preparation of the sensor was very simple and the reproducibility of preparation was very good. In particular, it offers possibilities for sensor miniaturization.

Journal Article↗

PubMed2001

Studies on the expression of mRNA for anion transport related proteins in corneal endothelial cells.

PURPOSE: Chloride and bicarbonate are necessary for maintenance of fluid transport by the corneal endothelium, however there is little information on the identity of anion transport proteins that could serve as anion efflux mechanisms in endothelial cells. Therefore, we ask whether mRNA for the anion transport related proteins, CFTR, CLC-2, ClC-3, ClC-5 and AE2, are expressed in human, bovine or rabbit corneal endothelium. METHODS: RT-PCR was performed for CFTR, CLC-2, ClC-3, ClC-5 and AE2 using total RNA from fresh human, bovine and rabbit corneal endothelium as well as cultured bovine corneal endothelial cells (CBCEC). Specificity of PCR products was confirmed by sequencing. RESULTS: RT-PCR analysis gave positive bands at the predicted size for CLC-3 and CLC-5 from fresh human, rabbit and bovine as well as CBCEC. However, for CLC-2, no band was apparent around the predicted size from fresh and cultured corneal endothelium. A band at the predicted size was obtained for CFTR from fresh human, rabbit and bovine endothelium, as well as from CBCEC. RT-PCR analysis for AE2 produced specific bands from fresh human, rabbit and bovine corneal endothelium, but no positive band was obtained from CBCEC. Sequencing analysis further confirmed the identities of CLC-3, CLC-5, CFTR and AE2 in corneal endothelium. CONCLUSIONS: CFTR, CLC-3 and ClC-5 are expressed in fresh and cultured corneal endothelial cells. However, consistent with previous immunoblots studies, AE2 is only expressed in fresh corneal endothelium. These results have implications for modeling possible apical anion efflux mechanisms in corneal endothelium.

Animals↗

PubMed2001

Effect of AQP1 expression level on Co(2) permeability in bovine corneal endothelium.

PURPOSE: Corneal endothelial fluid transport is dependent on HCO(3)(-) and CO(2) fluxes. CO(2) permeability (P:CO(2)) measurements in an oocyte expression system and in reconstituted proteoliposomes have suggested that the water channel AQP1 can transport CO(2). An AQP1 knockout mouse model, however, showed no evidence for CO(2) transport through AQP1 in erythrocytes or lung. Because HCO(3)(-) and CO(2) fluxes are essential to endothelial function, the current study was conducted to determine whether AQP1 expression levels in confluent cultures of bovine corneal endothelial cells (BCECs) affects membrane PCO(2). METHODS: BCEC endogenous AQP1 expression was reduced by antisense oligonucleotide (AO) transfection or adenoviral antisense-AQP1 (AV) infection. AQP1 was overexpressed by adenoviral sense-AQP1 (SV) infection, which directs expression of recombinant AQP1. RESULTS: Expression of AQP1 and osmotic water permeability (control P(f) = 0.046 +/- 0.005 cm/sec) were reduced 45% and 36.5%, respectively, by AO transfection and reduced 67% and 49%, respectively, by AV infection. SV infection induced a more than threefold overexpression of AQP1 but showed only a 37% increase in P(f). Adenoviral empty virus (EV) infection did not change AQP1 expression or P(f). PCO(2) was determined by measuring the rate of intracellular pH decrease after exposure to CO(2)/HCO(3)(-)-rich solutions, as measured by the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Apparent PCO(2) of BCEC (0.0036 +/- 0.00023 cm/sec) was not different among control, oligonucleotide-transfected, and adenoviral-infected cells. P(f) could also be reduced more than 50% by 3 to 5 minutes' exposure of control cells to 0.5 mM p-chloromercuriphenylsulfonic acid (pCMBS), but this had no effect on rates of intracellular pH decrease. CONCLUSIONS: AQP1 does not contribute to PCO(2) in corneal endothelial cells.

Adenoviridae↗

PubMed2001

Marek's disease virus latency.

MDV latency is defined as the persistence of the viral genome in the absence of production of infectious virus except during reactivation. A number of systems for studying MDV latency exist, and most involve the use of lymphoblastoid cells or tumors. It has been difficult to divorce latency and transformation. Understanding the relationship between these two states remains a major challenge for the MDV system. Based on their patterns of expression, the MDV LATs are apt to be important in the balance between latent and lytic infections. The LATs are a complex group of transcripts. The profile of gene expression that characterizes latency differs among all herpesviruses, and MDV is no exception. MDV LATs bear little resemblance to LATs of other alphaherpesviruses or to the LATs of other lymphotropic herpesviruses. LAT splicing patterns are complex and the relationships among various spliced species or between these species and the large 10-kb transcript are unknown. In addition, the existence of any protein gene products of significance is unknown at this time. More work is needed to further investigate the significance and function of these RNAs. Better technology to construct mutants in the MDV system is badly needed, since the analysis of mutants in the chicken is a powerful and unique advantage of the MDV system.

Animals↗

PubMed2001

mRNA and protein expression of Fas associated death domain protein in apoptosic hepatocyte induced by tumor necrosis factor-alpha.

OBJECTIVE: To study the expression of protein and mRNA of Fas associated death domain protein (FADD) in the apoptosic hepatocyte induced by D-galactosamine (GalN) and TNF-alpha. METHODS: Fulminant hepatic failure (FHF) was induced by injection of GalN into sensitized BALB/c mice by TNF-alpha. Hepatocyte apoptosis was examined by electric microscopy, in site end labeling (ISEL) and DNA electrophoresis on 1.5% agarose gel. The expression of protein and mRNA of FADD were detected by immunohistochemistry and PT-PCR. RESULTS: GalN/TNF-alpha may induce hepatocyte apoptosis, necrosis and liver failure. The expression of protein and mRNA of FADD were positively correlative with the apoptosic hepatocytes. CONCLUSION: TNF-alpha induces hepatocyte apoptosis by up regulation expression of FADD protein.

Adaptor Proteins, Signal Transducing↗

PubMed2001

[Influence of scatter factor/hepatocyte growth factor on the growth and transmission of hepatocarcinoma SMMC 7721 cells].

OBJECTIVE: To explore the influence of scatter factor/hepatocyte growth factor (SF/HGF) on the growth and transmission of hepatocarcinoma SMMC 7721 cells by SF/HGF cDNA transfection. METHODS: Gene transfection was operated by lipofectin method. In vitro, SF/HGF and c-met expression was tested by ELISA and Western blot. Cell proliferation and motility were compared by growth curves and wound healing assays. In vivo, cells before and after transfection were implanted subcutaneously into nude mice respectively to observe tumor growth and metastasis. RESULTS: After transfection, the expression of SF/HGF reached 694 pg/ml, compared with 0.26 pg/ml before transfection; however the expression of c-met protein did not change obviously. Growth curves showed that cells after transfection proliferated more rapidly than those before transfection and had an increased capability of motility together with enlarged morphological changes. In vivo experiment, tumors originated from SF/HGF(+) cells grew faster than those from SF/HGF(-) cells and had embolism formed inside and metastasis in the corresponding lung tissues, whereas no such findings in SF/HGF(-) cell originated tumors. CONCLUSIONS: High expression of SF/HGF can stimulate tumor cell proliferation and metastasis in hepatocellular carcinoma

Animals↗

PubMed2001

[Immune response induced by hepatitis B DNA vaccine pCI-S-IRES- ProT alpha].

OBJECTIVE: To investigate immune response in mice inoculated with DNA vaccine. METHODS: pCI-S and pCI-S-IRES- ProT alpha were constructed by gene technology, transferred into cell line NIH-3T3 mediated by lipofectamine. Mice were inoculated with these plasmids too. HBsAg and HBsAb were detected by ELISA, and transcriptions of these plasmids were detected by RT-PCR. RESULTS: pCI-S -IRES- ProT alpha, and pCI-S were effectively expressed in cultured cells and vaccinated animals. Humoral immune and specific T-cell proliferative responses were stronger in pCI-S-IRES group than pCI-S group. CONCLUSIONS: Plasmids coexpression of prothymosin alpha and hepatitis B surface antigen can improve immune responses significantly.

Animals↗

PubMed2000

Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development.

Auxin plays a key role in lateral root formation, but the signaling pathway for this process is poorly understood. We show here that NAC1, a new member of the NAC family, is induced by auxin and mediates auxin signaling to promote lateral root development. NAC1 is a transcription activator consisting of an N-terminal conserved NAC-domain that binds to DNA and a C-terminal activation domain. This factor activates the expression of two downstream auxin-responsive genes, DBP and AIR3. Transgenic plants expressing sense or antisense NAC1 cDNA show an increase or reduction of lateral roots, respectively. Finally, TIR1-induced lateral root development is blocked by expression of antisense NAC1 cDNA, and NAC1 overexpression can restore lateral root formation in the auxin-response mutant tir1, indicating that NAC1 acts downstream of TIR1.

Amino Acid Sequence↗

PubMed2000

Effect of tissue non-specific alkaline phosphatase in maintenance of structure of murine colon and stomach.

The gastrointestinal tract of mammals secretes a phospholipid-rich membrane that is enriched in alkaline phosphatase (AP) and surfactant proteins (surfactant-like particle, SLP). The production of this particle is stimulated in the small intestine by fat feeding and in cultured cells in vitro by transfection with intestinal alkaline phosphatase (IAP). To test whether tissue non-specific alkaline phosphatase (TNAP) was a factor in stimulating surfactant-like particle production in stomach and colon (tissues expressing TNAP), mice lacking this enzyme were studied. Mice were harvested at 8 days of life, when body weight of homozygous animals (TNAP -/-) was about half that of congenic controls (TNAP +/+) or heterozygotes (TNAP +/-), but before seizures had begun. No difference in content of the major SLP protein (65 kDa) by Western blotting or immunocytochemistry was seen in stomach or colon of TNAP -/- vs. TNAP +/+ animals, but the content was only about half in the IAP-expressing small bowel. Transmission electron microscopy of the TNAP -/- small bowel showed large dilated lysosomes and residual bodies. Colonocytes and gastric surface epithelial cells from the same animals showed mitochondria containing homogeneous dense inclusions, consistent with neutral lipid. In the underweight homozygous animals, there was a decrease in the neuronal content of submucosal ganglia in the jejunum and ileum and of myenteric ganglia in the jejunum of TNAP -/- animals. These findings suggest that (1) TNAP is not important in maintaining surfactant-like particle content of tissues that express TNAP, (2) normal fat absorption is important in maintaining SLP content in the small intestine, and (3) TNAP is important in the maintenance of some intestinal structures, and perhaps their function.

Alkaline Phosphatase↗

PubMed2000

Detection and analysis of Bacillus subtilis growth with piezoelectric quartz crystal impedance based on starch hydrolysis.

A piezoelectric quartz crystal (PQC) impedance method based on the alpha-amylase-catalyzed hydrolysis of starch present in a culture medium has been developed for in situ monitoring of the whole growth process of Bacillus subtilis and the variation in the activity of alpha-amylase during bacterial growth. An S-shaped response behavior was observed for Deltaf(0), and simultaneously inverse S-shaped responses were found for DeltaR(1) and DeltaL(1). The ratio of DeltaR(1) to Deltaf(0) or DeltaL(1) coincided well with that calculated from Martin's equations reflecting the solution density-viscosity effect, suggesting that the continuing change in liquid loading onto the PQC surface causes significant variation in Deltaf(0), DeltaR(1), and DeltaL(1). Bacterial growth equations were derived from the kinetics of the enzyme-catalyzed hydrolysis of starch, which fit well with the experimental responses of Deltaf(0), DeltaR(1), and DeltaL(1). Kinetic parameters of bacterial growth, including the asymptote (A), the maximum specific growth rate (microm), and the lag time (lambda), were obtained and were in good agreement with those obtained from the pour plate count method. The variation in the activity of alpha-amylase exhibited peak-type behavior with its maximum value at the later stage of the log phase. In addition, the influence of initial bacterial concentration was also investigated.

Algorithms↗

PubMed2000

Study of the Adsorption of Glutathione on a Gold Electrode by Using Electrochemical Quartz Crystal Impedance, Electrochemical Impedance Spectroscopy, and Cyclic Voltammetry.

Adsorption of a biological peptide, glutathione, on a gold electrode was studied by using electrochemical quartz crystal impedance (EQCI), electrochemical impedance spectroscopy (EIS), and cyclic voltammetry (CV) techniques. The time courses of responses of piezoelectric and electrochemical impedance parameters were simultaneously obtained during the adsorption processes of the two forms of peptide, oxidized and reduced glutathione. It was found that the frequency curve due to the oxidized glutathione (GSSG) adsorption exhibited a character of a sum of two exponential functions. For reduced glutathione (GSH), the frequency adsorption curve could be expressed by a first-order reaction kinetic model and the corresponding kinetic parameters at different amounts of GSH were obtained. The heterogeneous charge-transfer rate constants of ferricyanide/ferrocyanide before and after the peptide adsorption were determined by CV and EIS methods, respectively. According to the simple equivalent electric network of the electrochemical interface, the electrochemical impedance parameters were also obtained. The results showed that the proposed method should be found in wider applications in interfacial biochemistry studies since these combined techniques have advantages in real time multidimensional information including electrochemical and electrochemical impedance parameters. Copyright 2000 Academic Press.

Journal Article↗

PubMed2000

The two isozymes of rat intestinal alkaline phosphatase are products of two distinct genes.

Rat intestinal alkaline phosphatases (IAP-I and -II) differ in primary structure, substrate specificity, tissue localization, and response to fat feeding. This study identifies two distinct genes ( approximately 5-6 kb) corresponding to each isozyme and containing 11 exons of nearly identical size. The exon-intron junctions are identical with those found in IAP genes from other species. The 1.7 and 1.2 bp of 5' flanking regions isolated from each gene, respectively, contain Sp1 and gut-enriched Kruppel-like factor (GKLF) binding sites, but otherwise show little identity. There is a potential CAAT-box 14 bp 5' to the transcriptional start site, 36 bp upstream from IAP-I, and a TATA-box 31 bp 5' to the transcriptional start site, 55 bp upstream from IAP-II. Transfection of these promoter regions (linked to luciferase as a reporter gene) into a kidney cell line, COS-7, produced the differential response to oleic acid expected from in vivo studies, i.e., threefold increase using the 5' flanking region of IAP-II, but not IAP-I. This response was not reproduced by 5,8,11,14-eicosatetraynoic acid (ETYA) or clofibrate, suggesting that peroxisome proliferator response elements are not involved. Isolation of the IAP-II gene will allow determination of the sequences responsible for dietary fat response in the enterocyte.

5' Untranslated Regions↗

PubMed2000

Prenatal X-irradiation increases GFAP- and calbindin D28k-immunoreactivity in the medial subdivision of the nucleus of solitary tract in the rat.

Glial fibrillary acidic protein- (GFAP) and calbindin D28k-immunoreactivity (IR) were investigated in the medial subdivision of the nucleus of the solitary tract (mNST) of prenatally X-irradiated rats. Pregnant rats were exposed to a single whole-body X-irradiation on day 11 or 16 of gestation at a dose of 1. 3 Gy. The offspring were killed at 7-14 days of age for the immunohistochemical observations. Rat pups showed strong GFAP-IR at the level rostral to the obex when receiving X-rays on day 11 of gestation, with hypertrophy of astrocyte cell bodies and cytoplasmic processes, but weak GFAP-IR when receiving X-rays on day 16 of gestation. Calbindin D28k-IR was stronger in the animals receiving X-rays on day 11 or 16 of gestation compared to that in the control animals. In the present study, the increase of GFAP- and calbindin D28k-IR cells in the mNST might indicate that adaptative mechanisms are taking place to preserve integrated nervous system function and possibly, to provide neuroprotection.

Adaptation, Physiological↗

PubMed2000

Expression and localization of Na(+)-HCO(3)(-) cotransporter in bovine corneal endothelium.

Functional studies support the presence of the Na(+)-HCO(3)(-) cotransporter (NBC) in corneal endothelium and possibly corneal epithelium; however, molecular identification and membrane localization have not been reported. To test whether NBC is expressed in bovine cornea, Western blotting was performed, which showed a single band at approximately 130 kDa for freshly isolated and cultured endothelial cells, but no band for epithelium. Two isoforms of NBC have recently been cloned in kidney (kNBC) and pancreas (pNBC). RT-PCR was run using cultured and fresh bovine corneal endothelial and fresh corneal epithelial total RNA and specific primers for kNBC and pNBC. RT-PCR analysis for pNBC was positive in endothelium and weak in epithelium. The RT-PCR product was subcloned and confirmed as pNBC by sequencing. No specific bands for kNBC were obtained from corneal cells. Indirect immunofluorescence and confocal microscopy indicated that NBC locates predominantly to the basolateral membrane in corneal endothelial cells. Furthermore, Na(+)-dependent HCO(3)(-) fluxes and HCO(3)(-)-dependent cotransport with Na(+) were elicited only from the basolateral side of corneal endothelial cells. Therefore, we conclude that pNBC is present in the basolateral membrane of both fresh and cultured bovine corneal endothelium and weakly expressed in the corneal epithelium.

Amino Acid Sequence↗

PubMed2000

Selective impairment of corticotropin-releasing factor1 (CRF1) receptor-mediated function using CRF coupled to saporin.

CRF is the main component in the brain neuropeptide effector system responsible for the behavioral, endocrine, and physiological activation that accompanies stress activation. Reduced CRF system activation plays a role in the etiology of a variety of psychiatric and metabolic disease states. We have developed a novel protein conjugate that joins native rat/human CRF to a ribosome-inactivating protein, saporin (CRF-SAP), for the purpose of targeted inactivation of CRF receptor-expressing cells. Cytotoxicity measurements revealed that CRF-SAP (1-100 nM) produced concentration-dependent and progressive cell death over time in CRF1 receptor-transfected L cells, but at similar concentrations had no effect on CRF2alpha receptor-transfected cells. The CRF-SAP-induced toxicity in CRF1-transfected cells was prevented by coincubation with the competitive CRF1/CRF2 receptor peptide antagonist, [D-Phe12]CRF-(12-41), or the selective nonpeptide CRF1 receptor antagonist, NBI 27914. Finally, in cultured rat pituitary cells that express native CRF1 receptors, CRF-SAP suppressed CRF-induced (1 nM) ACTH release. GnRH (1-10 nM) stimulated LH release was also assessed in the same pituitary cultures. Although there was a slight decrease in LH release from these cultures, this decrease was observed with CRF-SAP or SAP alone, suggesting that the response was nonspecific. Taken together, these results suggest the utility of CRF-SAP as a specific and subtype-selective tool for long term impairment of CRF1 receptor-expressing cells.

Adrenocorticotropic Hormone↗